The goal of this project is to understand the regulation of HIV RNA splicing in terms of positive and negative cis elements within the viral genome and the role of cellular regulatory proteins which interact with these elements.The proposed studies will focus on the regulation by cellular factors of HIV RNA splicing in the regions including and flanking the first and second coding exons of the HIV tat (transactivator) and rev genes.The first specific aim is to define further cis-acting RNA splicing elements in the HIV first tat coding exon. The investigator will use in vitro splicing assays with HIV RNA substrates and analysis of HIV RNA in cells transiently transfected with mutant proviral clones to further define this element. The replication phenotype of virus containing mutations in he splicing element will also be studied. The second aim is to identify and characterize cellular RNA-binding factors interacting with the negative splicing regulatory element. Evidence for the presence of a factor or factors that bind to the negative splicing regulatory element in the first tat coding exon has been obtained. This factor(s) will be characterized and, if it a novel factor, the gene will be cloned and sequenced. The third goal is to define cis-acting positive and negative splicing elements in the second tat/rev coding exon. Elements will be defined using in vitro splicing assays and transient transfections.If novel factors bind to these elements, the genes will be cloned and sequenced.